Acoustic assessment of fluids in a plurality of reservoirs

ABSTRACT

The invention provides devices and methods for acoustically assessing the contents in a plurality of reservoirs. Each reservoir has a portion adapted to contain a fluid, and an acoustic radiation generator is positioned in acoustic coupling relationship to each of the reservoirs. Acoustic radiation generated by the acoustic radiation generator is transmitted through at least the portion of each reservoir to an analyzer. The analyzer is capable of analyzing a characteristic of the transmitted acoustic radiation and optionally correlating the characteristic to a property of the reservoirs&#39; contents. The invention is particularly suited for assessing the contents of a plurality of reservoirs to allow for accuracy and control over the dispensing of fluids therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.10/703,737, filed Nov. 6, 2003, which is a continuation-in-part ofapplication Ser. No. 09/964,212 filed Sep. 25, 2001, issued as U.S. Pat.No. 6,666,541, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/727,392, filed Nov. 29, 2000, now abandoned,which is a continuation-in-part of U.S. patent application Ser. No.09/669,996, filed Sep. 25, 2000, now abandoned. All the applicationsrecited in this paragraph are incorporated by reference herein.

TECHNICAL FIELD

The invention relates generally to the use of acoustic energy to assessthe contents of a plurality of reservoirs. In particular, the inventionrelates to devices and methods for acoustically assessing the contentsof a plurality of reservoirs in order to enhance accuracy and precisionin dispensing fluids from the reservoirs. The invention is particularlysuited for use in conjunction with combinatorial synthetic andanalytical systems that employ biomolecular libraries containing a largenumber of different fluid reservoirs.

BACKGROUND

The discovery of novel and useful materials depends largely on thecapacity to make and characterize new compositions of matter. As aresult, recent research relating to novel materials having usefulbiological, chemical, and/or physical properties has focused on thedevelopment and implementation of new methods and systems forsynthesizing and evaluating potentially useful chemical compounds. Inparticular, high-speed combinatorial methods have been developed toaddress the general need in the art for systematic, efficient, andeconomical material synthesis techniques as well as for methods toanalyze and to screen novel materials for useful properties.

Generally, it is important to control the quality of the startingmaterials in any chemical synthesis process. Otherwise, the integrity ofthe process and the quality of the resulting product would becompromised. Quality control of the starting materials is a particularlyimportant issue in combinatorial synthesis procedures. In suchprocedures, for example, those employed in peptide drug discoveryapplications, a large number of starting compounds may be dispensed in apredetermined sequence from a compound library to synthesize a batch ofa drug containing a specific peptide sequence. Should any of thestarting compounds contain an unacceptable level of a contaminant orexhibit an unacceptable degree of degradation, the resulting compoundmay be rendered useless. In effect, all starting compounds employed forthe batch synthesis would be wasted. This is particularly problematicwhen the one or more of the starting compounds are rare or expensive.

Similarly, combinatorial testing techniques may be employed inanalytical and testing procedures. For example, a plurality ofpharmacologically active candidate compounds may be delivered to a testsample in combination in order to assess whether synergistic effects areachieved. If any one of the candidate compounds is compromised inquality, however, the accuracy and reliability of the assessment may bereduced. Thus, further testing may be necessary, adding significantly tothe overall time and cost associated with the combinatorial testingprocess.

High-speed combinatorial methods often involve the use of arraytechnologies that require accurate dispensing of fluids each having aprecisely known chemical composition, concentration, stoichiometry,ratio of reagents, and/or volume. Such array technologies may beemployed to carry out various synthetic processes and evaluations. Arraytechnologies may employ large numbers of different fluids to form aplurality of reservoirs that, when arranged appropriately, createcombinatorial libraries. In order to carry out combinatorial techniques,a number of fluid dispensing techniques have been explored, such as pinspotting, pipetting, inkjet printing, and acoustic ejection. Many ofthese techniques possess inherent drawbacks that must be addressed,however, before the fluid dispensing accuracy required for thecombinatorial methods can be achieved. For instance, a number of fluiddispensing systems are constructed using networks of tubing or otherfluid-transporting vessels. Tubing, in particular, can entrap airbubbles, and nozzles may become clogged by lodged particulates. As aresult, system failure may occur and cause spurious results.Furthermore, cross-contamination between the reservoirs of compoundlibraries may occur due to inadequate flushing of tubing and pipettetips between fluid transfer events. Cross-contamination can easily leadto inaccurate and misleading results.

Acoustic ejection provides a number of advantages over otherfluid-dispensing technologies. In contrast to inkjet devices, nozzlelessfluid ejection devices are not subject to clogging and its associateddisadvantages, e.g., misdirected fluid or improperly sized droplets.Furthermore, acoustic technology does not require the use of tubing orinvolve invasive mechanical actions, for example, those associated withthe introduction of a pipette tip into a reservoir of fluid.

Acoustic ejection has been described in a number of patents. Forexample, U.S. Pat. No. 4,308,547 to Lovelady et al. describes a liquiddrop emitter that utilizes acoustic principles to eject droplets from abody of liquid onto a moving document to result in the formation ofcharacters or bar codes thereon. A nozzleless inkjet printing apparatusis used in which controlled drops of ink are propelled by an acousticforce produced by a curved transducer at or below the surface of theink. Similarly, U.S. patent application Ser. No. 09/964,212 describes adevice for acoustically ejecting a plurality of fluid droplets towarddiscrete sites on a substrate surface for deposition thereon. The deviceincludes an acoustic radiation generator that may be used to eject fluiddroplets from a reservoir, as well as to produce an acoustic wave fordetection that is transmitted to the fluid surface of the reservoir tobecome a reflected acoustic wave. Characteristics of the reflectedacoustic radiation may then be analyzed in order to assess the spatialrelationship between the acoustic radiation generator and the fluidsurface. Thus, acoustic ejection may provide an added advantage in thatthe proper use of acoustic radiation provides feedback relating to theprocess of acoustic ejection itself.

Regardless of the dispensing technique used, however, inventory andmaterials handling limitations generally dictate the capacity ofcombinatorial methods to synthesize and analyze increasing numbers ofsample materials. For instance, during the formatting and dispensingprocesses, microtiter plates that contain a plurality of fluids inindividual wells may be thawed, and the contents of selected wells thenextracted for use in a combinatorial method. When a pipetting system isemployed during extraction, a minimum loading volume may be required forthe system to function properly. Similarly, other fluid dispensingsystems may also require a certain minimum reservoir volume to functionproperly. Thus, for any fluid dispensing system, it is important tomonitor the reservoir contents to ensure that at least a minimum amountof fluid is provided. Such content monitoring generally serves todescribe the overall performance of a fluid dispensing system, as wellas to maintain the integrity of the combinatorial methods.

In addition, during combinatorial synthesis or analysis processes,environmental effects may play a role in altering the reservoircontents. For example, dimethylsulfoxide (DMSO) is a common organicsolvent employed to dissolve or suspend compounds commonly found in druglibraries. DMSO is highly hygroscopic and tends to absorb any ambientwater with which it comes into contact. The absorption of water dilutesthe concentration of the compounds in the DMSO as well as altering theability of the DMSO to suspend the compounds. Furthermore, theabsorption of water may promote the decomposition of water-sensitivecompounds.

A number of patents describe the use of acoustic energy to assess thecontents of a container. U.S. Pat. No. 5,507,178 to Dam, for example,describes a sensor for determining the presence of a liquid and foridentifying the type of liquid in a container. The ultrasonic sensordetermines the presence of the liquid through an ultrasonic “liquidpresence sensing means” and identifies the type of liquid through a“liquid type identification means” that includes a pair of electrodesand an electrical pulse generating means. This device suffers from thedisadvantage that the sensor must be placed in contact with the liquid.

U.S. Pat. No. 5,880,364 to Dam, on the other hand, describes anon-contact ultrasonic system for measuring the volume of liquid in aplurality of containers. An ultrasonic sensor is disposed opposite thetop of the containers. A narrow beam of ultrasonic radiation istransmitted from the sensor to the open top of an opposing container tobe reflected from the air-liquid interface of the container back to thesensor. By using the round trip transit time of the radiation and thedimensions of the containers being measured, the volume of liquid in thecontainer can be calculated. This device cannot be used to assess thecontents of sealed containers. In addition, the device lacks precisionbecause air is a poor conductor of acoustic energy. Thus, while thisdevice may provide rough estimate of the volume of liquid in relativelylarge containers, it is unsuitable for use in providing a detailedassessment of the contents of reservoirs typically used withcombinatorial techniques. In particular, this device cannot determinethe position of the bottom of containers since substantially all of theemitted acoustic energy is reflected from the liquid surface and doesnot penetrate to the bottom. Small volume reservoirs such as microtiterplates are regular arrays of fluid containers. The location of thebottoms of the containers in such arrays can vary by a significantfraction of the nominal height of a container due to bow in the plate.Thus, detection of the position of the liquid surface only leads tosignificant errors in height and thus volume estimation in commoncontainers.

Thus, there is a need in the art for improved methods and devices thatare capable of monitoring the contents of a plurality of reservoirs, acapability that is particularly useful in synthetic and analyticalprocesses to increase the robustness, efficiency, and effectiveness ofthe combinatorial techniques employed therein.

SUMMARY OF THE INVENTION

In a first embodiment, the invention provides a device for acousticallyassessing the contents of a plurality of fluid reservoirs eachcomprising a solid surface, wherein a portion of each reservoir isadapted to contain a fluid. The device also includes an acousticradiation generator with a means for positioning the generator in anacoustically coupled relationship to each reservoir, such that theacoustic radiation generated is transmitted through the solid surfaceand the portion of each reservoir adapted to contain a fluid. Ananalyzer positioned to receive the acoustic radiation is provided foranalyzing a characteristic of the transmitted acoustic radiation.

Typically, the inventive device includes a single acoustic radiationgenerator and a plurality of removable reservoirs. In addition, theacoustic radiation generator may comprise a component common to theanalyzer, such as a piezoelectric element. Optionally, the acousticgenerator may represent a component of an acoustic ejector, which ejectsdroplets from the reservoirs. In such a case, the device may furthercomprise a means to focus the acoustic radiation.

The analyzer may be adapted to analyze acoustic radiation, therebypermitting determination of the fluid volume contained in eachreservoir. In addition or in the alternative, the analyzer may also beadapted to analyze acoustic radiation to determine a specific propertyof the fluid contained in each reservoir. The specific fluid propertymay be, for example, viscosity, surface tension, acoustic impedance,acoustic attenuation, solid content, or impurity content.

In another embodiment, the invention relates to a method foracoustically assessing the contents of one or more fluid reservoirs. Themethod involves selecting a reservoir from a plurality of reservoirseach comprising a solid surface, wherein a portion of each reservoir isadapted to contain a fluid, and positioning an acoustic radiationgenerator in an acoustically coupled relationship to the selectedreservoir. Once positioned, the acoustic radiation generator is actuatedso that generated acoustic radiation is transmitted through the solidsurface and through the portion of the selected reservoir adapted tocontain a fluid and to an analyzer capable of analyzing a characteristicof the transmitted radiation. The analyzer is then operated to analyzethe characteristic of the transmitted radiation in order to assess thecontents of the selected reservoir. Optionally, the acoustic radiationgenerator could be repositioned to permit assessment of the contents ofthe remaining reservoirs.

Typically, the contents of the selected reservoir are assessed bydetermining the difference in the acoustic radiation before and aftertransmission through the reservoir. The results of the acoustic analysismay be stored electronically for later use.

In a further embodiment, the invention relates to a method foraccurately dispensing fluid from one or more reservoirs. The methodinvolves positioning an acoustic radiation generator in an acousticallycoupled relationship to a reservoir selected from a plurality ofreservoirs, in order to transmit acoustic radiation through at least aportion of the selected reservoir adapted to contain a fluid. Acharacteristic of the transmitted acoustic radiation is then analyzed inorder to assess the reservoir's contents. Once the acoustic radiationcharacteristic has been assessed by the acoustic radiation analysis,fluid is dispensed accordingly from the selected reservoir, preferablyby acoustic means.

In yet a further embodiment, the invention relates to a device fordispensing a fluid from a plurality of reservoirs each having a portionadapted to contain a fluid. Improvement in the device is accomplished byproviding an acoustic radiation generator, a means for positioning theacoustic radiation generator in an acoustically coupled relationship toeach reservoir such that acoustic radiation is transmitted through atleast the portion of each reservoir, and an analyzer for analyzing theacoustic radiation. The analyzer is positioned to receive thetransmitted acoustic radiation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B, collectively referred to as FIG. 1, schematicallyillustrate in simplified cross-sectional view a preferred embodiment ofthe invention that allows both the acoustic assessment in reflectivemode of the contents of a plurality of reservoirs and the ejection offluid droplets therefrom. As depicted, the device comprises first andsecond reservoirs, a combined acoustic analyzer and ejector, and anejector positioning means. FIG. 1A shows the acoustic ejectoracoustically coupled to the first reservoir; the ejector is activated inorder to eject a droplet of fluid from within the first reservoir towarda site on a substrate surface to form an array. FIG. 1B shows theacoustic ejector acoustically coupled to a second reservoir.

FIG. 2 schematically illustrates in simplified cross-sectional view anembodiment of the inventive device designed to permit acousticalassessment of the contents of a plurality of reservoirs in transmissivemode.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific fluids,biomolecules, or device structures, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a reservoir” includes a plurality of reservoirs, referenceto “a fluid” includes a plurality of fluids, reference to “abiomolecule” includes a combination of biomolecules, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set forthbelow.

The terms “acoustic coupling” and “acoustically coupled” as used hereinrefer to a state wherein an object is placed in direct or indirectcontact with another object so as to allow acoustic radiation to betransferred between the objects without substantial loss of acousticenergy. When two entities are indirectly acoustically coupled, an“acoustic coupling medium” is needed to provide an intermediary throughwhich acoustic radiation may be transmitted. Thus, an ejector may beacoustically coupled to a fluid, such as by immersing the ejector in thefluid, or by interposing an acoustic coupling medium between the ejectorand the fluid, in order to transfer acoustic radiation generated by theejector through the acoustic coupling medium and into the fluid.

The term “attached,” as in, for example, a substrate surface having amoiety “attached” thereto, includes covalent and noncovalent binding,adsorption, and physical immobilization. The terms “binding” and “bound”are identical in meaning to the term “attached.”

The term “array” as used herein refers to a two-dimensional arrangementof features, such as an arrangement of reservoirs (e.g., wells in a wellplate) or an arrangement of different moieties, including ionic,metallic, or covalent crystalline, e.g., molecular crystalline,composite or ceramic, glassine, amorphous, fluidic or molecularmaterials on a substrate surface (as in an oligonucleotide or peptidicarray). Arrays are generally comprised of regular, ordered features, asin, for example, a rectilinear grid, parallel stripes, spirals, and thelike, but non-ordered arrays may be advantageously used as well. Anarray is distinguished from the more general term “pattern” in thatpatterns do not necessarily contain regular and ordered features.

The terms “biomolecule” and “biological molecule” are usedinterchangeably herein to refer to any organic molecule that is, was, orcan be a part of a living organism, regardless of whether the moleculeis naturally occurring, recombinantly produced, or chemicallysynthesized in whole or in part. The terms encompass, for example,nucleotides, amino acids, and monosaccharides, as well as oligomeric andpolymeric species, such as oligonucleotides and polynucleotides,peptidic molecules, such as oligopeptides, polypeptides and proteins,saccharides such as disaccharides, oligosaccharides, polysaccharides,mucopolysaccharides or peptidoglycans (peptido-polysaccharides) and thelike. The terms also encompass ribosomes, enzyme cofactors,pharmacologically active agents, and the like. Additional informationrelating to the term “biomolecule” can be found in U.S. Ser. No.09/964,212.

The term “fluid” as used herein refers to matter that is nonsolid, or atleast partially gaseous and/or liquid, but not entirely gaseous. A fluidmay contain a solid that is minimally, partially, or fully solvated,dispersed, or suspended. Examples of fluids include, without limitation,aqueous liquids (including water per se and salt water) and nonaqueousliquids such as organic solvents and the like. As used herein, the term“fluid” is not synonymous with the term “ink” in that an ink mustcontain a colorant and may not be gaseous.

The terms “focusing means” and “acoustic focusing means” refer to ameans for causing acoustic waves to converge at a focal point, either bya device separate from the acoustic energy source that acts like anoptical lens, or by the spatial arrangement of acoustic energy sourcesto effect convergence of acoustic energy at a focal point byconstructive and destructive interference. A focusing means may be assimple as a solid member having a curved surface, or it may includecomplex structures such as those found in Fresnel lenses, which employdiffraction in order to direct acoustic radiation. Suitable focusingmeans also include phased arrays as are known in the art and described,for example, in U.S. Pat. No. 5,798,779 to Nakayasu et al. and Amemiyaet al. (1997) Proceedings of the 1997 IS&T NIP 13 InternationalConference on Digital Printing Technologies, pp. 698-702.

The terms “library” and “combinatorial library” are used interchangeablyherein to refer to a plurality of chemical or biological moietiesarranged in a pattern or an array such that the moieties areindividually addressable. In some instances, the plurality of chemicalor biological moieties is present on the surface of a substrate, and inother instances, the plurality of moieties represents the contents of aplurality of reservoirs. Preferably, but not necessarily, each moiety isdifferent from each of the other moieties. The moieties may be, forexample, peptidic molecules and/or oligonucleotides.

The term “moiety” refers to any particular composition of matter, e.g.,a molecular fragment, an intact molecule (including a monomericmolecule, an oligomeric molecule, and a polymer), or a mixture ofmaterials (for example, an alloy or a laminate).

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

The term “reservoir” as used herein refers to a receptacle or chamberfor containing a fluid. In some instances, a fluid contained in areservoir necessarily will have a free surface, e.g., a surface thatallows acoustic radiation to be reflected therefrom or a surface fromwhich a droplet may be acoustically ejected. A reservoir may also be alocus on a substrate surface within which a fluid is constrained.

The term “substrate” as used herein refers to any material having asurface onto which one or more fluids may be deposited. The substratemay be constructed in any of a number of forms including, for example,wafers, slides, well plates, or membranes. In addition, the substratemay be porous or nonporous as required for deposition of a particularfluid. Suitable substrate materials include, but are not limited to,supports that are typically used for solid phase chemical synthesis,such as polymeric materials (e.g., polystyrene, polyvinyl acetate,polyvinyl chloride, polyvinyl pyrrolidone, polyacrylonitrile,polyacrylamide, polymethyl methacrylate, polytetrafluoroethylene,polyethylene, polypropylene, polyvinylidene fluoride, polycarbonate, anddivinylbenzene styrene-based polymers), agarose (e.g., Sepharose®),dextran (e.g., Sephadex®), cellulosic polymers and otherpolysaccharides, silica and silica-based materials, glass (particularlycontrolled pore glass, or “CPG”), functionalized glasses, ceramics, andsuch substrates treated with surface coatings, e.g., with microporouspolymers (particularly cellulosic polymers such as nitrocellulose),microporous metallic compounds (particularly microporous aluminum),antibody-binding proteins (available from Pierce Chemical Co., RockfordIll.), bisphenol A polycarbonate, or the like. Additional informationrelating to the term “substrate” can be found in U.S. patent applicationSer. No. 09/964,212.

The invention accordingly relates to devices and methods foracoustically assessing the contents of a plurality of fluid reservoirs.An embodiment of the invention is a device which includes a plurality ofreservoirs, each adapted to contain a fluid, and an acoustic radiationgenerator for generating acoustic radiation. The inventive device alsoincludes a means for positioning the acoustic radiation generator in anacoustically coupled relationship to each reservoir such that theacoustic radiation generated by the acoustic radiation generator istransmitted through at least a portion of each reservoir. An analyzerfor analyzing a characteristic of acoustic radiation is positioned toreceive the transmitted acoustic radiation.

The device may be constructed to include the reservoirs as an integratedor permanently attached component of the device. However, to providemodularity and interchangeability of components, it is preferred thatdevice be constructed with removable reservoirs. Generally, thereservoirs are arranged in a pattern or an array to provide eachreservoir with individual systematic addressability. In addition, whileeach of the reservoirs may be provided as a discrete or stand-aloneitem, in circumstances that require a large number of reservoirs, it ispreferred that the reservoirs be attached to each other or representintegrated portions of a single reservoir unit. For example, thereservoirs may represent individual wells in a well plate. Many wellplates suitable for use with the device are commercially available andmay contain, for example, 96, 384, 1536, or 3456 wells per well plate.Manufactures of suitable well plates for use in the employed deviceinclude Corning, Inc. (Corning, N.Y.) and Greiner America, Inc. (LakeMary, Fla.). However, the availability of such commercially availablewell plates does not preclude the manufacture and use of custom-madewell plates containing at least about 10,000 wells, or as many as100,000 to 500,000 wells, or more.

Furthermore, the material used in the construction of reservoirs must becompatible with the fluids contained therein. Thus, if it is intendedthat the reservoirs or wells contain an organic solvent such asacetonitrile, polymers that dissolve or swell in acetonitrile would beunsuitable for use in forming the reservoirs or well plates. Similarly,reservoirs or wells intended to contain DMSO must be compatible withDMSO. For water-based fluids, a number of materials are suitable for theconstruction of reservoirs and include, but are not limited to, ceramicssuch as silicon oxide and aluminum oxide, metals such as stainless steeland platinum, and polymers such as polyester andpolytetrafluoroethylene. For fluids that are photosensitive, thereservoirs may be constructed from an optically opaque material that hassufficient acoustic transparency for substantially unimpairedfunctioning of the device.

In addition, to reduce the amount of movement and time needed to alignthe acoustic radiation generator with each reservoir or reservoir wellduring operation, discussed infra, it is preferable that the center ofeach reservoir be located not more than about 1 centimeter, preferablynot more than about 1 millimeter, and optimally not more than about 0.5millimeter, from a neighboring reservoir center. These dimensions tendto limit the size of the reservoirs to a maximum volume. The reservoirsare constructed to contain typically no more than about 1 mL, preferablyno more than about 1 μL, and optimally no more than about 1 nL, offluid. To facilitate handling of multiple reservoirs, it is alsopreferred that the reservoirs be substantially acousticallyindistinguishable.

Generally, a single acoustic radiation generator is employed, though aplurality of acoustic radiation generators may be employed as well. Allacoustic radiation generators employ a vibrational element or transducerto generate acoustic radiation. Often, a piezoelectric element isemployed to convert electrical energy into mechanical energy associatedwith acoustic radiation. When a single acoustic radiation generator isemployed, the positioning means should allow for the acoustic radiationgenerator to move from one reservoir to another quickly and in acontrolled manner, thereby allowing fast and controlled scanning of thecontents of the reservoirs. In order to ensure optimal performance, itis important to keep in mind that there are two basic kinds of motion:pulse and continuous. Pulse motion involves the discrete steps of movingan acoustic radiation generator into position, keeping it stationarywhile it emits acoustic energy, and moving the generator to the nextposition; again, using a high performance positioning means with such amethod allows repeatable and controlled acoustic coupling at eachreservoir in less than 0.1 second. Typically, the pulse width is veryshort and may enable over 10 Hz reservoir transitions, and even over1000 Hz reservoir transitions. A continuous motion design, on the otherhand, moves the acoustic radiation generator and the reservoirscontinuously, although not at the same speed.

In some instances, the analyzer is positioned in fixed alignment withrespect to the acoustic radiation generator. In other instances,however, a means similar to that described above is provided foraltering the relative positions of the analyzer with respect to thereservoirs. The relative position of the analyzer and the acousticradiation generator depends on the particular configuration of thedevice. In some instances, the device may be configured to operate intransmissive mode, such that the generated radiation is transmittedthrough the entirety of the reservoir whose contents are assessed. Insuch a case, the reservoir may be interposed between the acousticradiation generator and an acoustic analyzer. As another option, thedevice may be configured to operate in a reflective mode, such that theacoustic radiation is transmitted only through a portion the reservoirwhose contents are being assessed. In such a case, the analyzer may bepositioned in a manner appropriate for this configuration, e.g., inorder to receive reflected acoustic radiation. In any case, the acousticradiation generator should be positioned such that generated acousticradiation is transmitted through the portion of each reservoir mostlikely to contain a fluid for optimal performance. This reduces thechance that the analyzer will erroneously determine that a reservoir isempty. For example, as fluids ordinarily flow to the bottom ofcontainers or are driven there by centrifugation; the acoustic radiationgenerator should be positioned such that generated acoustic radiationcan be transmitted through the bottom of a reservoir.

In a preferred configuration, as discussed in detail below, the analyzeris positioned to receive acoustic radiation reflected from a freesurface of a fluid contained in each reservoir. In such a configuration,the acoustic radiation generator may comprise one or more componentsshared with the analyzer. The components common to the acousticradiation generator and the analyzer may include a vibrational elementthat converts one form of energy into another, e.g., a piezoelectricelement that converts between acoustic/mechanical energy and electricalenergy.

The analyzer may be constructed to perform a number of functions. Forexample, the analyzer may be adapted to analyze acoustic radiation todetermine the volume of fluid in each reservoir. In addition, or in thealterative, the analyzer may be adapted to analyze acoustic radiation todetermine a property of fluid in each reservoir. Fluid properties thatcan be determined in this manner include, but are not limited to,viscosity, surface tension, acoustic impedance, acoustic attenuation,solid content, and impurity content.

Thus, the invention also provides a method for acoustically assessingthe contents of one or more reservoirs. The method involves providing aplurality of reservoirs, each reservoir adapted to contain a fluid, andpositioning an acoustic radiation generator in acoustic couplingrelationship to a selected reservoir. Once positioned, the acousticradiation generator is actuated to generate acoustic radiation that istransmitted through at least a portion of the selected reservoir to ananalyzer. The analyzer is then used to analyze acoustic radiation thathas been transmitted through at least a portion of the selectedreservoir, thereby assessing the contents of the selected reservoir.Optionally, the acoustic radiation generator may be repositioned toallow for the assessment of the contents of the remaining reservoirs aswell.

As discussed above, the reservoirs may be constructed to reduce theamount of movement and time needed to align the acoustic radiationgenerator with each reservoir or reservoir well during operation. As ageneral matter of convenience and efficiency, it is desirable to analyzean entire library of different moieties in a relatively short amount oftime, e.g., about one minute. Thus, the inventive method typicallyallows for the analysis of the contents of the reservoirs at a rate ofat least about 96 reservoirs per minute. Faster analysis rates of atleast about 384, 1536, and 3456 reservoirs per minute are achievablewith present-day technology as well. Thus, the invention can be operatedto analyze in one minute the contents of each well of most (if not all)well plates that are currently commercially available. Properimplementation of the inventive method should yield a reservoir analysisrate of at least about 10,000 reservoirs per minute. Currentcommercially available positioning technology allows the acousticradiation generator to be moved from one reservoir to another, withrepeatable and controlled acoustic coupling at each reservoir, in lessthan about 0.1 second for high performance positioning means and in lessthan about 1 second for ordinary positioning means. A custom designedsystem will allow the acoustic radiation generator to be moved from onereservoir to another with repeatable and controlled acoustic coupling inless than about 0.001 second.

By analyzing acoustic radiation that has been transmitted through atleast a portion of a selected reservoir, one may accurately determinethe contents of the selected reservoir. For example, the assessment mayinvolve determining the volume of fluid in the reservoir or determininga property of the fluid in the reservoir. As discussed above, fluidproperties that may be determined include, but are not limited to,viscosity, surface tension, acoustic impedance, solid content, andimpurity content. In some instances, the assessment may involvemeasuring the travel time of acoustic radiation through the reservoir.In addition, or in the alternative, the assessment may involvedetermining the difference of acoustic radiation before and aftertransmission through the reservoir. For temperature-dependentproperties, a temperature measurement means known in art, such asthermocouples, may be used in conjunction with such analyses.Optionally, the results of acoustic analysis performed by the acousticanalyzer may be stored. Thus, the inventive device may include, forexample, a storage means comprising rewritable and/or permanent datastorage media for storing the results of acoustic analysis performed bythe analyzer.

Acoustic assessment as described above may be employed to improve fluiddispensing from each of a plurality of reservoirs adapted to contain afluid. Thus, another embodiment of the invention relates to a device fordispensing fluid from each of a plurality of reservoirs adapted tocontain a fluid. This device may include any of a number of knowntechniques for dispensing fluids involving contact-based fluiddispensing, e.g., pin spotting, pipetting, and inkjet printing, ornon-contact based fluid dispensing, e.g., acoustic ejection. However,the inventive device represents a novel and nonobvious improvement overthe fluid dispensing devices known in the art since it provides forenhanced accuracy and precision in fluid dispensing through the use of ameans for acoustically assessing the contents of the reservoirs. Themeans for acoustically assessing the contents of the reservoirs issimilar to the previously described device for assessing the contents ofa plurality of fluid reservoirs in that it also comprises an acousticradiation generator for generating acoustic radiation and an analyzerfor analyzing a characteristic of acoustic radiation. A means forpositioning the acoustic radiation generator in acoustic couplingrelationship to each reservoir is used to ensure that acoustic radiationgenerated by the acoustic radiation generator is transmitted through atleast a portion of each reservoir. Furthermore, the analyzer ispositioned to receive the transmitted acoustic radiation.

As discussed above, acoustic ejection provides a number of advantagesover other fluid dispensing technologies. In addition, compatibleacoustic ejection technology described in U.S. Ser. No. 09/964,212involves an ejector comprising an acoustic radiation generator forgenerating acoustic radiation and a focusing means for focusing theacoustic radiation generated at a focal point within and sufficientlynear the fluid surface in each of a plurality of reservoirs to result inthe ejection of droplets therefrom. Thus, the invention also provides adevice that can carry out both acoustic ejection and assessment. In sucha case, the acoustic radiation generator may serve as a component ofboth an acoustic ejector and an acoustic assessing means.

Optionally, a focusing means is typically provided for focusing theacoustic radiation generated by the acoustic generator. In the presentinvention, any of a variety of focusing means may be employed inconjunction with the acoustic generator in order to eject droplets froma reservoir through the use of focused acoustic radiation. For example,one or more curved surfaces may be used to direct acoustic radiation toa focal point near a fluid surface. One such technique is described inU.S. Pat. No. 4,308,547 to Lovelady et al. Focusing means with a curvedsurface have been incorporated into the construction of commerciallyavailable acoustic transducers such as those manufactured by PanametricsInc. (Waltham, Mass.). In addition, Fresnel lenses are known in the artfor directing acoustic energy at a predetermined focal distance from anobject plane. See, e.g., U.S. Pat. No. 5,041,849 to Quate et al. Fresnellenses may have a radial phase profile that diffracts a substantialportion of acoustic energy into a predetermined diffraction order atdiffraction angles that vary radially with respect to the lens. Thediffraction angles should be selected to focus the acoustic energywithin the diffraction order on a desired object plane. Optimally, thedevice is adapted to eject fluid from a reservoir according to theresults of acoustic analysis performed by the analyzer.

The device may also provide certain performance-enhancingfunctionalities. For example, the device may include a means forcontrolling the temperature of one or more of the reservoirs. Suchtemperature controlling means may be employed in the inventive device toimprove the accuracy of measurement and may be employed regardless ofwhether the device includes a fluid dispensing functionality. In thecase of aqueous fluids, the temperature controlling means should havethe capacity to maintain the reservoirs at a temperature above about 0°C. In addition, the temperature controlling means may be adapted tolower the temperature in the reservoirs. Such temperature lowering maybe required because repeated application of acoustic energy to areservoir of fluid may result in heating of the fluid. Such heating canresult in unwanted changes in fluid properties such as viscosity,surface tension and density. Design and construction of such temperaturecontrolling means are known to one of ordinary skill in the art and maycomprise, e.g., components such a heating element, a cooling element, ora combination thereof. For many biomolecular applications, reservoirs offluids are stored frozen and thawed for use. During use, it is generallydesired that the fluid containing the biomolecule be kept at a constanttemperature, with deviations of no more than about 1° C. or 2° C.therefrom. In addition, for a biomolecular fluid that is particularlyheat sensitive, it is preferred that the fluid be kept at a temperaturethat does not exceed about 10° C. above the melting point of the fluid,preferably at a temperature that does not exceed about 5° C. above themelting point of the fluid. Thus, for example, when thebiomolecule-containing fluid is aqueous, it may be optimal to keep thefluid at about 4° C. during ejection.

Moreover, the device may be adapted to assess and/or dispense fluids ofvirtually any type and amount desired. The fluid may be aqueous and/ornonaqueous. Examples of fluids include, but are not limited to, aqueousfluids including water per se and water-solvated ionic and non-ionicsolutions, organic solvents, lipidic liquids, suspensions of immisciblefluids, and suspensions or slurries of solids in liquids. Because theinvention is readily adapted for use with high temperatures, fluids suchas liquid metals, ceramic materials, and glasses may be used; see, e.g.,co-pending patent application U.S. Ser. No. 09/669/194 (“Method andApparatus for Generating Droplets of Immiscible Fluids”), inventorsEllson and Mutz, filed on Sep. 25, 2000, and assigned to Picoliter, Inc.(Sunnyvale, Calif.). Furthermore, because of the precision that ispossible using the inventive technology, the device may be used to ejectdroplets from a reservoir adapted to contain no more than about 100nanoliters of fluid, preferably no more than 10 nanoliters of fluid. Incertain cases, the ejector may be adapted to eject a droplet from areservoir adapted to contain about 1 to about 100 nanoliters of fluid.This is particularly useful when the fluid to be ejected contains rareor expensive biomolecules, wherein it may be desirable to eject dropletshaving a volume of about 1 picoliter or less, e.g., having a volume inthe range of about 0.025 pL to about 1 pL.

Thus, another embodiment of the invention relates to a method fordispensing fluid from one or more reservoirs. Once an acoustic radiationgenerator is positioned, in acoustic coupling relation to a reservoirselected from a plurality of reservoirs, acoustic radiation generated bythe acoustic radiation generator may be transmitted through at least aportion of the selected reservoir. The acoustic radiation is thenanalyzed in order to assess the contents of the reservoir, and fluid isdispensed from the selected reservoir according to the assessment.Typically, the fluid is dispensed through acoustic ejection, though theinventive method may employ contact-based fluid dispensing either as analternative to or as a supplement to noncontact-based fluid dispensing.Optionally, the above process may be repeated for additional reservoirs.

It should be noted that there are a number of different ways to combineacoustic assessment with fluid dispensing, depending on the intendedpurpose of the combination. As discussed above, fluid may be dispensedfrom a reservoir after the contents of the reservoir are acousticallyassessed. This allows an operator to fine tune the dispensing accordingto the condition of the contents of the reservoir. In addition, fluidmay be dispensed from a reservoir before the contents of the reservoirare acoustically assessed. In such a case, acoustic assessment may serveto confirm the quality of fluid dispensation as well as to ensure thatthe dispensing process does not unexpectedly alter the contents of thereservoir. For example, by assessing the volume of fluid remaining in areservoir after a fluid has been dispensed from the reservoir, anoperator may determine the quantity of fluid actually removed from thereservoir. In some instances, acoustic assessment and fluid dispensationmay occur simultaneously.

FIG. 1 illustrates a preferred embodiment of the inventive device insimplified cross-sectional view. In this embodiment, the inventivedevice allows for acoustic assessment of the contents of a plurality ofreservoirs as well as acoustic ejection of fluid droplets from thereservoirs. The inventive device is shown in operation to form abiomolecular array bound to a substrate. As with all figures referencedherein, in which like parts are referenced by like numerals, FIG. 1 isnot to scale, and certain dimensions may be exaggerated for clarity ofpresentation. The device 11 includes a plurality of reservoirs, i.e., atleast two reservoirs, with a first reservoir indicated at 13 and asecond reservoir indicated at 15. Each is adapted to contain a fluidhaving a fluid surface. As shown, the first reservoir 13 contains afirst fluid 14 and the second reservoir 15 contains a second fluid 16.Fluids 14 and 16 each have a fluid surface respectively indicated at 17and 19. Fluids 14 and 16 may the same or different. As shown, thereservoirs are of substantially identical construction so as to besubstantially acoustically indistinguishable, but identical constructionis not a requirement. The reservoirs are shown as separate removablecomponents but may, as discussed above, be fixed within a plate or othersubstrate. For example, the plurality of reservoirs may compriseindividual wells in a well plate, optimally although not necessarilyarranged in an array. Each of the reservoirs 13 and 15 is preferablyaxially symmetric as shown, having vertical walls 21 and 23 extendingupward from circular reservoir bases 25 and 27 and terminating atopenings 29 and 31, respectively, although other reservoir shapes may beused. The material and thickness of each reservoir base should be suchthat acoustic radiation may be transmitted therethrough and into thefluid contained within the reservoirs.

The device also includes an acoustic ejector 33 comprised of an acousticradiation generator 35 for generating acoustic radiation and a focusingmeans 37 for focusing the acoustic radiation at a focal point within thefluid from which a droplet is to be ejected, near the fluid surface. Theacoustic radiation generator contains a transducer 36, e.g., apiezoelectric element, commonly shared by an analyzer. As shown, acombination unit 38 is provided that both serves as a controller and acomponent of an analyzer. Operating as a controller, the combinationunit 38 provides the piezoelectric element 36 with electrical energythat is converted into mechanical and acoustic energy. Operating as acomponent of an analyzer, the combination unit receives and analyzeselectrical signals from the transducer. The electrical signals areproduced as a result of the absorption and conversion of mechanical andacoustic energy by the transducer.

As shown in FIG. 1, the focusing means 37 may comprise a single solidpiece having a concave surface 39 for focusing acoustic radiation, butthe focusing means may be constructed in other ways as discussed below.The acoustic ejector 33 is thus adapted to generate and focus acousticradiation so as to eject a droplet of fluid from each of the fluidsurfaces 17 and 19 when acoustically coupled to reservoirs 13 and 15,and thus to fluids 14 and 16, respectively. The acoustic radiationgenerator 35 and the focusing means 37 may function as a single unitcontrolled by a single controller, or they may be independentlycontrolled, depending on the desired performance of the device.Typically, single-ejector designs are preferred over multiple-ejectordesigns because accuracy of droplet placement and consistency in dropletsize and velocity are more easily achieved with a single ejector.

There are also a number of ways to acoustically couple the ejector 33 toeach individual reservoir and thus to the fluid therein. One suchapproach is through direct contact as is described, for example, in U.S.Pat. No. 4,308,547 to Lovelady et al., wherein a focusing meansconstructed from a hemispherical crystal having segmented electrodes issubmerged in a liquid to be ejected. The aforementioned patent furtherdiscloses that the focusing means may be positioned at or below thesurface of the liquid. However, this approach for acoustically couplingthe focusing means to a fluid is undesirable when the ejector is used toeject different fluids in a plurality of containers or reservoirs, asrepeated cleaning of the focusing means would be required in order toavoid cross-contamination. The cleaning process would necessarilylengthen the transition time between each droplet ejection event. Inaddition, in such a method, fluid would adhere to the ejector as it isremoved from each container, wasting material that may be costly orrare.

Thus, a preferred approach would be to acoustically couple the ejectorto the reservoirs and reservoir fluids without contacting any portion ofthe ejector, e.g., the focusing means, with any of the fluids to beejected. To this end, the present invention provides an ejectorpositioning means for positioning the ejector in controlled andrepeatable acoustic coupling with each of the fluids in the reservoirsto eject droplets therefrom without submerging the ejector therein. Thistypically involves direct or indirect contact between the ejector andthe external surface of each reservoir. When direct contact is used inorder to acoustically couple the ejector to each reservoir, it ispreferred that the direct contact is wholly conformal to ensureefficient acoustic energy transfer. That is, the ejector and thereservoir should have corresponding surfaces adapted for mating contact.Thus, if acoustic coupling is achieved between the ejector and reservoirthrough the focusing means, it is desirable for the reservoir to have anoutside surface that corresponds to the surface profile of the focusingmeans. Without conformal contact, efficiency and accuracy of acousticenergy transfer may be compromised. In addition, since many focusingmeans have a curved surface, the direct contact approach may necessitatethe use of reservoirs having a specially formed inverse surface.

Optimally, acoustic coupling is achieved between the ejector and each ofthe reservoirs through indirect contact, as illustrated in FIG. 1A. Inthis figure, an acoustic coupling medium 41 is placed between theejector 33 and the base 25 of reservoir 13, with the ejector andreservoir located at a predetermined distance from each other. Theacoustic coupling medium may be an acoustic coupling fluid, preferablyan acoustically homogeneous material in conformal contact with both theacoustic focusing means 37 and each reservoir. In addition, it ispreferable to ensure that the fluid medium is substantially free ofmaterial having different acoustic properties than the fluid mediumitself. Furthermore, it is preferred that the acoustic coupling mediumis comprised of a material having acoustic properties that facilitatethe transmission of acoustic radiation without significant attenuationin acoustic pressure and intensity. Also, the acoustic impedance of thecoupling medium should facilitate the transfer of energy from thecoupling medium into the container. As shown, the first reservoir 13 isacoustically coupled to the acoustic focusing means 37, such that anacoustic wave is generated by the acoustic radiation generator anddirected by the focusing means 37 into the acoustic coupling medium 41,which then transmits the acoustic radiation into the reservoir 13.

In operation, reservoirs 13 and 15 are each filled with first and secondfluids 14 and 16, respectively, as shown in FIG. 1. The acoustic ejector33 is positionable by means of ejector positioning means 43, shown belowreservoir 13, in order to achieve acoustic coupling between the ejectorand the reservoir through acoustic coupling medium 41. Once the ejector,the reservoir, and the substrate are in proper alignment, the acousticradiation generator 35 is activated to produce acoustic radiation thatis directed toward a free fluid surface 17 of the first reservoir. Theacoustic radiation will then travel in a generally upward directiontoward the free fluid surface 17. The acoustic radiation will bereflected under different circumstances. Typically, reflection willoccur when there is a change in the acoustic property of the mediumthrough which the acoustic radiation is transmitted. It has beenobserved that a portion of the acoustic radiation traveling upward willbe reflected from by the reservoir bases 25 and 27 as well as the freesurfaces 17 and 19 of the fluids contained in the reservoirs 13 and 15.

As discussed above, acoustic radiation may be employed for use as ananalytical tool as well as to eject droplets from a reservoir. In ananalytical mode, the acoustic radiation generator is typically activatedso as to generate low energy acoustic radiation that is insufficientlyenergetic to eject a droplet from the fluid surface. This is typicallydone by using a short pulse (on the order of tens of nanoseconds), whichis extremely short compared to that required for droplet ejection (onthe order of microseconds). By determining the time it takes for theacoustic radiation to be reflected by the fluid surface back to theacoustic radiation generator, and then correlating that time with thespeed of sound in the fluid, the distance—and thus the fluid height—maybe calculated. Of course, care must be taken in order to ensure thatacoustic radiation reflected by the interface between the reservoir baseand the fluid is discounted.

Thus, the present invention represents a significant improvement overknown technologies relating to the acoustic assessment of the contentsof a plurality of reservoirs. As discussed above, prior acousticassessment of the contents of liquid reservoirs typically involvedplacing a sensor in direct contact with the liquid. This means that thesensor must be cleaned between each use to avoid cross-contamination ofthe contents of the reservoirs. In contrast, the invention allows forassessment of the contents of a plurality of containers without directcontact with the contents of the containers.

While other non-contact acoustic systems are known in the art, suchsystems provide only an indirect and approximate assessment of thecontents of a reservoir. For example, the acoustic system described inU.S. Pat. No. 5,880,364 to Dam employs a technique in which the acousticradiation is transmitted from a sensor through an air-containing portionof the container and then reflected from the air-liquid interface of thecontainer back to the sensor. The round trip transit time is used todetermine the volume of the air-containing portion of the container. Thevolume of liquid in the container is determined by subtracting thevolume of the container not occupied by the liquid from the volume ofthe entire container. One drawback of this technique is that it cannotprovide an accurate assessment of the liquid volume in a container whenthe volume of the container is not precisely known. This is particularlyproblematic when small reservoirs such as those typically used incombinatorial techniques are employed. The dimensional variability forsuch containers is relatively large when considered in view of the smallvolume of the reservoirs. Furthermore, the technique cannot be employedwhen the volume of the container is completely unknown or alterable.Finally, since acoustic radiation never penetrates the liquid, thereflected radiation can at best only provide information relating to thesurface of the liquid, not information relating to the bulk of theliquid.

In contrast, because the invention involves the transmission of acousticradiation through the portion of each reservoir adapted to contain afluid, the transmitted acoustic radiation may provide informationrelating to the volume as well as the properties of the fluids in thereservoir. For example, the invention provides a plurality ofreservoirs, wherein a portion of each reservoir is adapted to contain afluid. A fluid contained in a reservoir must ordinarily contact a solidsurface of the reservoir. When the invention is employed in a reflectivemode, some of the generated acoustic radiation may be reflected byinterface between the fluid and the solid surface, while the remainderis transmitted through a fluid contained in the reservoir. Thetransmitted radiation is then reflected by another surface, e.g., a freesurface, of the fluid contained in the reservoir. By determining thedifference in round-trip transit time between the two portions, thevolume of the fluid in the reservoir may be accurately determined. Inaddition, transmission of acoustic radiation through the fluid allowscharacteristics of the acoustic radiation to be altered by fluid. Thus,information relating to a property of the fluid may be deduced byanalyzing a characteristic of the transmitted acoustic radiation.

In addition, air, like other gases, exhibits low acoustic impedance, andacoustic radiation tends to attenuate more in gaseous materials than inliquid or solid materials. For example, the attenuation at 1 MHz for airis approximately 10 dB/cm while that of water is 0.002 dB/cm. Since theacoustic system described in U.S. Pat. No. 5,880,364 to Dam requiresacoustic radiation to travel through air, this system requires much moreenergy to operate. Thus, the present invention represents a more energyefficient technology that may be employed to provide more accurate anddetailed assessment of the contents of a plurality of fluid reservoirs.Some of this additional accuracy can be achieved by using higherfrequency acoustic waves (and hence shorter wavelengths) as theseacoustic waves can be transmitted effectively through liquids yet wouldbe very rapidly attenuated in air.

It will be appreciated by those of ordinary skill in the art thatconventional or modified sonar techniques may be employed. Thus, theacoustic radiation will be reflected back at the piezoelectric element36, where the acoustic energy will be converted into electrical energyfor analysis. The analysis may be used, for example, to reveal whetherthe reservoir contains any fluid at all. If fluid is present in thereservoir, the location and the orientation of the free fluid surfacewithin the reservoir may be determined, as well as the overall volume ofthe fluid. Characteristics of the reflected acoustic radiation may beanalyzed in order to assess the spatial relationship between theacoustic radiation generator and the fluid surface, the spatialrelationship between a solid surface of the reservoir and the fluidsurface, as well as to determine a property of the fluid in eachreservoir, e.g., viscosity, surface tension, acoustic impedance,acoustic attenuation, solid content, and impurity content. Once theanalysis has been performed, a decision may be made as to whether and/orhow to dispense fluid from the reservoir.

Depending on the type of assessment to be carried out, varioustechniques known in the art may be adapted for use in the presentinvention. Generally, interfacial energy measurements are routinelycarried out using contact-angle measurement. The present invention maybe adapted to perform such contact-angle measurements. In addition, anumber of other acoustic assessment techniques are known in the art. Forexample, U.S. Pat. No. 4,391,129 to Trinh described a system formonitoring the physical characteristics of fluids. The physicalcharacteristic may be determined from acoustic assessment of theinterfacial tension of fluids to a high degree of accuracy. U.S. Pat.No. 4,558,589 to Hemmes describes an ultrasonic blood-coagulationmonitor. U.S. Pat. No. 5,056,357 to Dymling et al. described acousticmethods for measuring properties in fluids through Doppler shifts. Otheracoustic assessment techniques that may be adapted for use in thepresent invention are described, for example, in U.S. Pat. Nos.4,901,245, 5,255,564, 5,410,518, 5,471,872, 5,533,402, 5,594,165,5,623,095, 5,739,432, 5,767,407, 5,793,705, 5,804,698, 6,119,510,6,227,040, and 6,298,726.

In order to form a biomolecular array on a substrate using the inventivedevice, substrate 45 is positioned above and in proximity to the firstreservoir 13 such that one surface of the substrate, shown in FIG. 1 asunderside surface 51, faces the reservoir and is substantially parallelto the surface 17 of the fluid 14 therein. Once the ejector, thereservoir, and the substrate are in proper alignment, the acousticradiation generator 35 is activated to produce acoustic radiation thatis directed by the focusing means 37 to a focal point 47 near the fluidsurface 17 of the first reservoir. That is, an ejection acoustic wavehaving a focal point near the fluid surface is generated in order toeject at least one droplet of the fluid, wherein the optimum intensityand directionality of the ejection acoustic wave is determined using theaforementioned analysis, optionally in combination with additional data.The “optimum” intensity and directionality are generally selected toproduce droplets of consistent size and velocity. For example, thedesired intensity and directionality of the ejection acoustic wave maybe determined by using the data from the above-described assessmentrelating to reservoir volume or fluid property data, as well asgeometric data associated with the reservoir. In addition, the data mayshow the need to reposition the ejector so as to reposition the acousticradiation generator with respect to the fluid surface, in order toensure that the focal point of the ejection acoustic wave is near thefluid surface, where desired. For example, if analysis reveals that theacoustic radiation generator is positioned such that the ejectionacoustic wave cannot be focused near the fluid surface, the acousticradiation generator is repositioned using vertical, horizontal, and/orrotational movement to allow appropriate focusing of the ejectionacoustic wave.

As a result, droplet 49 is ejected from the fluid surface 17 onto adesignated site on the underside surface 51 of the substrate. Theejected droplet may be retained on the substrate surface by solidifyingthereon after contact; in such an embodiment, it may be necessary tomaintain the substrate at a low temperature, i.e., a temperature thatresults in droplet solidification after contact. Alternatively, or inaddition, a molecular moiety within the droplet attaches to thesubstrate surface after contract, through adsorption, physicalimmobilization, or covalent binding.

Then, as shown in FIG. 1B, a substrate positioning means 50 repositionsthe substrate 45 over reservoir 15 in order to receive a droplettherefrom at a second designated site. FIG. 1B also shows that theejector 33 has been repositioned by the ejector positioning means 43below reservoir 15 and in acoustically coupled relationship thereto byvirtue of acoustic coupling medium 41. Once properly aligned, theacoustic radiation generator 35 of ejector 33 is activated to producelow energy acoustic radiation to assess the contents of the reservoir 15and to determine whether and/or how to eject fluid from the reservoir.Historical droplet ejection data associated with the ejection sequencemay be employed as well. Again, there may be a need to reposition theejector so as to reposition the acoustic radiation generator withrespect to the fluid surface, in order to ensure that the focal point ofthe ejection acoustic wave is near the fluid surface, where desired.Should the results of the assessment indicate that fluid may bedispensed from the reservoir, focusing means 37 is employed to directhigher energy acoustic radiation to a focal point 48 within fluid 16near the fluid surface 19, thereby ejecting droplet 53 onto thesubstrate 45.

It will be appreciated that various components of the device may requireindividual control or synchronization to form an array on a substrate.For example, the ejector positioning means may be adapted to ejectdroplets from each reservoir in a predetermined sequence associated withan array to be prepared on a substrate surface. Similarly, the substratepositioning means for positioning the substrate surface with respect tothe ejector may be adapted to position the substrate surface to receivedroplets in a pattern or array thereon. Either or both positioningmeans, i.e., the ejector positioning means and the substrate positioningmeans, may be constructed from, for example, motors, levers, pulleys,gears, a combination thereof, or other electromechanical or mechanicalmeans known to one of ordinary skill in the art. It is preferable toensure that there is a correspondence between the movement of thesubstrate, the movement of the ejector, and the activation of theejector to ensure proper array formation.

Accordingly, the invention relates to the assessment of the contents ofa plurality of reservoirs as well as to dispensing a plurality of fluidsfrom reservoirs, e.g., in order to form a pattern or an array, on thesubstrate surface 51. However, there are a number of different ways inwhich content assessment and fluid dispensing may relate to each other.That is, a number of different sequences may be employed for assessingthe contents of the reservoirs and for dispensing fluids therefrom. Insome instances, the contents of a plurality of reservoirs may beassessed before fluid is dispensed from any of the reservoirs. In otherinstances, the contents of each reservoir may be assessed immediatelybefore fluid is dispensed therefrom. The sequence used typically dependson the particular fluid-dispensing technique employed as well as theintended purpose of the sequence.

FIG. 2 illustrates an example of the inventive device that provides forassessment of the contents of a plurality of reservoirs in transmissivemode rather than in reflective mode. Considerations for the design andconstruction of this device are similar to those discussed above. Thus,the device 11 includes a first reservoir 13 and a second reservoir 15,each adapted to contain a fluid indicated at 14 and 16, respectively,and each of substantially identical construction. The first reservoir 13is depicted in an open state, while the second reservoir is depicted ina sealed state. An acoustic radiation generator 35 is positioned belowthe reservoirs, and analyzer 38 is positioned in opposing relationshipwith the acoustic radiation generator 35 above the reservoirs.

In operation, the contents of each of the reservoirs are acousticallyevaluated before pipette 60 is employed to dispense fluid therefrom. Asshown, the contents 14 of the first reservoir 13 have already beenacoustically assessed. As the assessment has revealed that the firstreservoir 13 contains at least a minimum acceptable level of fluid 14,the first reservoir 13 is open and ready for fluid to be dispensedtherefrom via pipette 60. The contents 16 of the second reservoir 15 areundergoing acoustic assessment, as depicted by FIG. 2, as the secondreservoir 15 is interposed between the acoustic radiation generator 35and the analyzer 38. The acoustic radiation generator 35 and theanalyzer 38 are acoustically coupled to the second reservoir viacoupling media 41 and 42, respectively. Once the acoustic radiationgenerator 35, the second reservoir 15, and the analyzer 38 are in properalignment, the acoustic radiation generator 35 is activated to produceacoustic radiation that is transmitted through the reservoir 15 and itscontents 16 toward the analyzer 38. The received acoustic radiation isanalyzed by an analyzer 38 as described above

It should be evident, then, that the invention provides a number ofpreviously unrealized advantages for assessing the contents of aplurality of reservoirs. First, acoustic assessment is a generallynoninvasive technique that may be carried out regardless of whether thereservoirs are sealed or open. That is, acoustic assessment does notrequire extracting a sample for analysis or other mechanical contactthat may result in sample cross-contamination. In addition, unlikeoptical detection techniques, optically translucent or transparentreservoirs are not required. This, of course, provides a wider range ofchoices for material that may be employed for reservoir construction. Inaddition, the use of opaque material would be particularly advantageousin instances wherein the reservoirs are constructed to containphotosensitive fluids.

Thus, variations of the present invention will be apparent to those ofordinary skill in the art. For example, while FIG. 1 depicts theinventive device in operation to form a biomolecular array bound to asubstrate, the device may be operated in a similar manner to format aplurality of fluids, e.g., to transfer fluids from odd-sized bulkcontainers to wells of a standardized well plate. Similarly, while FIG.2 illustrates that the acoustic radiation generator and the detector arein vertical opposing relationship, other spatial and/or geometricarrangements may be employed so long as acoustic radiation generated istransmitted through at least a portion of the reservoir to the detector.

As another example, the invention may be employed to detect whether thecontents of a sealed reservoir are at least partially frozen withoutopening the reservoir. This would be useful when it is known that areservoir contains a substance that is capable of existing as a fluidover a temperature range, but it is unclear what the temperature historyof the reservoir has been, e.g., whether it has been subjected tofreeze-thaw cycles. For example, water is capable of existing as a fluidat a temperature of about 0° C. to about 100° C. If it is unclearwhether the exterior temperature of a reservoir is indicative of thereservoir's interior temperature, but the reservoir is known to containliquid water, the inventive device is well suited to determine whetherany or all of the contents of the reservoir is a fluid.

In addition, the invention may be constructed as to be highly compatiblewith existing infrastructure of materials discovery and with existingautomation systems for materials handling. For example, the inventionmay be adapted for use as an alternative or a supplement to contentassessment means that are based on optical detection. In some instances,sonic markers may be provided in the reservoirs to identify the contentsof the reservoir. Thus, the invention may be employed as a means forinventory identification and control in a number of contexts, including,but not limited, to biological, biochemical, and chemical discovery andanalysis.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages, and modifications will beapparent to those skilled in the art to which the invention pertains.

All patents, patent applications, journal articles, and other referencescited herein are incorporated by reference in their entireties.

1. A device for acoustically determining a property of the contents of aplurality of fluid reservoirs, comprising: a plurality of reservoirseach comprising a solid surface, wherein a portion of each reservoir isadapted to contain a fluid; an acoustic radiation generator forgenerating acoustic radiation; a means for positioning the acousticradiation generator in acoustic coupling relationship to each reservoirsuch that acoustic radiation generated by the acoustic radiationgenerator is transmitted through the solid surface and the portion ofeach reservoir adapted to contain a fluid; and an analyzer for analyzinga characteristic of the transmitted acoustic radiation.
 2. The device ofclaim 1, comprised of a single acoustic radiation generator.
 3. Thedevice of claim 1, wherein the plurality of reservoirs are removablefrom the device.
 4. The device of claim 1, wherein the plurality ofreservoirs are individual wells in a well plate.
 5. The device of claim1, wherein the plurality of reservoirs are substantially acousticallyindistinguishable.
 6. The device of claim 1, wherein the plurality ofreservoirs are sealed.
 7. The device of claim 1, wherein the devicecomprises 96 reservoirs.
 8. The device of claim 7, wherein the devicecomprises 384 reservoirs.
 9. The device of claim 8, wherein the devicecomprises 1536 reservoirs.
 10. The device of claim 9, wherein the devicecomprises 10,000 reservoirs.
 11. The device of claim 10, wherein thedevice comprises 100,000 reservoirs.
 12. The device of claim 1, whereinat least one reservoir is constructed to contain no more than about 100nL of fluid.
 13. The device of claim 12, wherein at least one reservoiris constructed to contain no more than about 10 nL of fluid.
 14. Thedevice of claim 1, wherein at least one reservoir contains a fluid. 15.The device of claim 14, wherein the at least one reservoir contains anaqueous fluid.
 16. The device of claim 14, wherein the at least one ofthe reservoirs reservoir contains a nonaqueous fluid.
 17. The device ofclaim 16, wherein the nonaqueous fluid comprises an organic solvent. 18.The device of claim 14, wherein the fluid contains a biomolecule. 19.The device of claim 14, wherein the fluid is at least partially frozen.20. The device of claim 1, wherein at least one reservoir contains asubstance capable of existing as a fluid at a temperature of about 0° C.to about 100° C.
 21. The device of claim 1, further comprising a meansfor altering the relative position of the analyzer with respect to theplurality of reservoirs.
 22. The device of claim 1, wherein the analyzeris positioned to receive acoustic radiation reflected from a freesurface of a fluid contained in a reservoir.
 23. The device of claim 1,wherein the analyzer is adapted to analyze a characteristic of acousticradiation to determine the volume of fluid in each reservoir.
 24. Thedevice of claim 1, wherein the analyzer is adapted to analyze acharacteristic of acoustic radiation to determine a property of thefluid in each reservoir.
 25. The device of claim 24, wherein theproperty is height.
 26. The device of claim 24, wherein the property issurface orientation.
 27. The device of claim 1, wherein the acousticgenerator represents a component of an acoustic ejector for ejectingdroplets from the plurality of reservoirs.
 28. The device of claim 27,further comprising a focusing means for focusing the acoustic radiationgenerated by the acoustic generator.
 29. The device of claim 28, whereinthe focusing means is adapted to focus the acoustic radiation accordingto the results of acoustic analysis performed by the analyzer.
 30. Thedevice of claim 1, further comprising a storage means for storing theresults of acoustic analysis performed by the analyzer.
 31. The deviceof claim 30, wherein the storage means comprises rewritable data storagemedia.
 32. The device of claim 30, wherein the storage means comprisespermanent data storage media.
 33. The device of claim 30, furthercomprising the results of acoustic analysis performed by the analyzerstored in the storage means.
 34. The device of claim 1, furthercomprising a temperature control means for controlling the temperatureof the plurality of reservoirs.
 35. A method for acousticallydetermining a property of the contents of one or more reservoirs,comprising the steps of: (a) selecting a reservoir from a plurality ofreservoirs each comprising a solid surface, wherein a portion of eachreservoir is adapted to contain a fluid; (b) positioning an acousticradiation generator in acoustic coupling relationship to the selectedreservoir; (c) actuating the acoustic radiation generator to generateacoustic radiation so that the generated acoustic radiation is thentransmitted through the solid surface and through the portion of theselected reservoir adapted to contain a fluid to an analyzer capable ofanalyzing a characteristic of the transmitted radiation, thecharacteristic corresponding to a property of the contents of theselected reservoir; and (d) operating the analyzer to analyze thecharacteristic of the transmitted acoustic radiation.
 36. The method ofclaim 35, further comprising repeating steps (b), (c), and (d) for theremaining reservoirs.
 37. The method of claim 35, further comprising,after step (a), step (a′) dispensing a quantity of fluid from thereservoir.
 38. The method of claim 37, wherein step (a′) is carried outthrough acoustic ejection.
 39. The method of claim 37, wherein step (a′)is carried out after sufficient time has passed to allow for thecontents of the reservoir to melt.
 40. The method of claim 35, whereinstep (b) comprises positioning the acoustic radiation generator suchthat acoustic radiation generated by the acoustic generator is directedtoward a free surface of a fluid within the reservoir.
 41. The method ofclaim 35, further comprising, after step (d), step (e) correlating thecharacteristic to the volume of the contents in the reservoir.
 42. Themethod of claim 35, further comprising, after step (d), step (e)correlating the characteristic to a property of the contents in thereservoir.
 43. The method of claim 42, wherein the property is height.44. The method of claim 42, wherein the property is surface orientation.45. The method of claim 35, wherein step (d) comprises measuring thetravel time of the transmitted acoustic radiation through the reservoir.46. The method of claim 35, further comprising (e) storing the resultsof acoustic analysis performed by the acoustic analyzer.
 47. The methodof claim 35, wherein each of steps (a), (b), (c), and (d) is carried outwhile the reservoir is in a sealed state.
 48. A method for accuratelydispensing fluid from a reservoir, comprising the steps of: (a)positioning an acoustic radiation generator in acoustic couplingrelationship to a reservoir selected from a plurality of reservoirs,wherein a portion of each reservoir is adapted to contain a fluid; (b)transmitting acoustic radiation generated by the acoustic radiationgenerator through at least the portion of the selected reservoir adaptedto contain a fluid; (c) analyzing a characteristic of the transmittedacoustic radiation; and (d) dispensing fluid from the selected reservoiraccording to the analysis of the characteristic of the transmittedacoustic radiation.
 49. The method of claim 48, wherein step (d) iscarried out through acoustic ejection.
 50. The method of claim 48,wherein steps (a), (b), (c), and (d) are repeated for another reservoirselected from the plurality of reservoirs.
 51. In a device fordispensing one or more fluids from a plurality of reservoirs each havinga portion adapted to contain a fluid, the improvement comprisesproviding: an acoustic radiation generator for generating acousticradiation; a means for positioning the acoustic radiation generator inacoustic coupling relationship to each reservoir such that acousticradiation generated by the acoustic radiation generator is transmittedthrough at least the portion of each reservoir adapted to contain afluid; and an analyzer for analyzing a characteristic of acousticradiation.
 52. The device of claim 51, wherein the acoustic radiationgenerator represents a component of an acoustic ejector.